HCV is a major public health problem, infecting more than 170 million people worldwide. Most cases of HCV infection become persistent and may eventually lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped virus classified in the Flaviviridae family. The single-strand, positive-sense viral RNA genome encodes a single polyprotein precursor that is processed into three structural proteins (C, E1, E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) by host and viral proteases. The RNA genome of HCV has significant heterogeneity with six major genotypes and numerous subtypes. HCV infection is currently treated with alpha interferon (IFN&#945;)-based therapy. Although the treatment outcome is variable among the six major HCV genotypes, only 50% of treated patients infected with genotype 1 respond to therapy. The ability of HCV to establish persistent infection with great success in human has been attributed, in part, to a variety of strategies to evade host immune and IFN-induced defenses. Epidemiological studies suggest that up to 20% of acutely infected HCV patients can resolve the infection without treatment, which implies that innate and/or adaptive immune responses are indeed capable of controlling the outcome of HCV infection. The molecular mechanisms that regulate innate intracellular antiviral responses may therefore serve as pivotal points of control, potentially limiting host permissiveness for HCV replication and favorably modulating subsequent adaptive immune responses. A major consequence of virus infection is the induction of the type I interferons (IFN) including IFN&#945;/&#946;, which are cytokines of immune system with essential function in innate and adaptive immune response. Secreted IFN then triggers hundreds of IFN stimulated genes (ISGs) expression which establishes an antiviral state in the surrounding of virus-infected cells to limit viral replication, spread and infection. The in-vivo induction of interferons and interferon stimulated genes is a critical component of the human innate immune response to hepatitis C virus (HCV) and other viral pathogens. As a down-stream effector in the Type I interferon pathway, interferon stimulated gene 15 (ISG15) has been shown to play a pivotal role in this chain of innate immunity. ISG15 is a 15 KDa ubiquitin like protein. Upon IFN&#945; stimulation, ISG15 is induced and conjugated with target proteins, leading to a cascade of ubiquitin-like protein degradation. During infection with Ebola virus, ISG15 was found to suppress Nedd4-mediated ubiquitin E3 activity, which led to the inhibition of Ebola egress. Also, ISG15 dependent ISGylation has been shown to block the release of retrovirus from infected cells. Over-expression of ISG15 in human medulloblastoma cells can up-regulate IRF3-induced interferon response and inhibit the replication of Japanese encephalitis virus. Recent studies suggest that ISG15 participates in the host defense against viral infection through multiple pathways in addition to those involving interferon. The mechanisms of ISG-15 action in HCV infection are not clearly defined and controversial. In the past year, we employed short-term (siRNA) and stable (shRNA) inhibition of ISG15 in adapted human hepatoma cells (Huh 7.5.1) and measured effects on HCV replication and HCV egress into supernatant fluid. After treatment with siRNA ISG15 or shRNA ISg15, the level of ISG15 mRNA was reduced by 50% and 90%, respectively. Importantly, ISG15 silencing had only minor effects on reducing HCV intracellular RNA copy number, but reduced the level of HCV RNA in culture supernatants by 4-fold using siRNA and 8-fold using shRNA. The opposite and expected effect was observed when ISG15 was over-expressed, in which case HCV copy number in culture supernatants increased 3-fold. In examining the mechanism of reduced HCV egress following ISG15 silencing, it was shown that ISG15 knockdown reduced HCV IRES activation and the expression of viral proteins, particularly the core protein. We are still investigating how ISGylation might affect the association of HCV with lipid droplets. HCV is a positive-strand RNA virus. After released into cytoplasm, HCV RNA severs as a template for the viral translation and replication. Therefore, the coupling between the viral translation and viral RNA replication plays an important role in the modulation of HCV replication. In order to escape the host defense surveillance, HCV developed a complex strategy to coordinate these two processes. Of these, host factors have been documented to involve in this modulation. NF-kB, as a critical host transcription factor, has been implicated in the regulation of HCV infection and replication. The NF-kB family is composed of five structural similar proteins: p50, p52, RelA, c-Rel and RelB. In general, upon extracellular stimulation, RelA can rapidly translocate into nucleus through the phosphorylation of IkB and immediately induces the extensive gene expression. In viral infection, NF-kB has been shown to bind Alu repeats residing on chromosomes, leading to long-distance chromosomal interactions and initiating transcription of IFN- during the earlier phrases of viral infection. Therefore, in order to subvert innate immune responses, many viruses manage their distinctive viral proteins to target NF-kB mediated IFN- response. For instance, Influenza virus NS1 protein can suppress virus-induced NF-kB activation and IFN- synthesis. Vaccinia virus can also employ E3 protein to inhibit NF-kB-mediated IFN- induction. Some viruses can hijack cellular NF-kB signaling as their implements to antagonize IFN- and enhance viral transcription such as HIV-1, and EBV. To further clarify the role of NF-kB in the HCV lifecycle, we used shRNA RelA to knockdown the activation of NF-kB. Intriguingly, we found that RelA silencing significantly increased HCV IRES mediated viral translation, which in turn led to a higher level of viral protein expression in HCV infected cells. Furthermore, RelA silencing greatly enhanced HCV viral RNA replication inside the cells and the production of infectious HCV. Our studies showed that these enhancements were dependent on NF-kB mediated interferon beta response.